The present invention relates generally to the fields of developmental biology and molecular biology. More particularly, it concerns a striated muscle RING finger protein (MURF) involved in microtubule and intermediate filament stabilization of striated muscle cells.
The RING-finger is an unusual type of Cys-His zinc-binding motif found in a growing number of proteins with roles in signal transduction, gene transcription, differentiation, and morphogenesis (Borden, 1998; Saurin et al., 1996). A RING-B-box-coiled-coil (RBCC) subclass of RING-finger proteins contains an N-terminal RING-finger followed by a single or multiple additional zinc-finger domains, termed B-boxes, and a leucine-rich coiled-coil domain (Borden, 1998). The tripartite organization of these domains is evolutionarily conserved, suggesting an integrated and functional role for this overall protein structure. It should also be noted that the RING-finger and B-box motifs have been identified based on sequence homologies and are predicted to function as zinc-binding domains. However, their precise functions have not been fully elucidated. There is evidence suggesting that the RING-finger, B-box and coiled-coil domains mediate protein-protein interactions.
Several RBCC proteins have been implicated in oncogenesis. The RBCC member PML becomes fused to the retinoic acid receptor alpha in acute promyelocytic leukemia (De The et al., 1991). Similarly, the RBCC proteins BRCA1, Cb1, Rfp, TIF1, and MDM2 have been demonstrated to be oncogenic when fused to other factors through chromosomal translocation events (Saurin et al., 1996). Other RBCC proteins have been implicated in signal transduction, organellar biogenesis, chromosomal dynamics, viral pathogenesis, transcription, and developmental patterning (Saurin et al., 1996).
Recently, a complex congenital human disease, Opitz G/BBB syndrome, was shown to result from mutations in the RBCC protein, Mid1 (Quaderi et al., 1997). Opitz G/BBB syndrome is characterized by abnormalities of midline structures, including hypertelorism, clefts of lip and palate, larygotracheoesophageal defects, hypospadias, imperforate anus, and developmental delay. The Mid1 gene product is widely expressed during development and interacts with microtubules throughout the cell cycle (Cainarca et al., 1999). Overexpression of Mid1 leads to a stable population of microtubules resistant to depolymerization (Schweiger et al., 1999). Interestingly, mutations of Mid1 that are linked to Opitz G/BBB syndrome severely diminish the ability of Mid1 to interact with microtubules, suggesting that Mid1-microtubule interaction and/or microtubule dynamics are involved in the processes required for normal development of the midline structures affected in Opitz G/BBB syndrome.
Many questions remain regarding the function of Mid1-type proteins and their interactions with microtbules. Nonetheless, it is clear that such molecules play an important role in development, function and pathology of a wide variety of cell types.
Therefore, in one aspect of the invention, there is provided a DNA segment encoding a MURF-1, MURF-2 or MURF-3 polypeptide. The MURF-1, MURF-2 or MURF-3 polypeptide may be human, mouse, dog, rabbit, rat, Drosphila, yeast or other species. In a particular embodiment, the MURF-1 polypeptide has the sequence of SEQ ID NO:2, the MURF-2 polypeptide has the sequence of SEQ ID NO:4, and the MURF-3 polypeptide has the sequence of SEQ ID NO:6. In yet more particular embodiments, the MURF-1 DNA segment has the sequence of SEQ ID NO:1, the MURF-2 DNA segment has the sequence of SEQ ID NO:3, and the MURF-3 DNA segment has the sequence of SEQ ID NO:5.
The DNA segment may be positioned under the control of a promoter, for example, a promoter not native to the MURF-1, MURF-2 or MURF-3 coding region. The MURF-1, MURF-2 or MURF-3 coding region gene may be positioned in reverse orientation to the promoter, thereby capable of expressing an antisense product. The DNA segment may further comprise a polyadenylation signal. The DNA segment may further comprise an origin of replication. The DNA segment may be viral vector or a non-viral vector.
In another aspect of the invention, there is provided a host cell comprising a DNA segment that encodes a MURF-1, MURF-2 or MURF-3 polypeptide, wherein said DNA segment comprises a promoter heterologous to the MURF-1, MURF-2 or MURF-3 coding region. The host cell may further be defined as a prokaryotic host cell or a eukaryotic host cell. The host cell may be a secretory cell.
In yet another aspect of the invention, there is provided a method of using a host cell comprising an expression cassette comprising a polynucleotide encoding a MURF-1, MURF-2 or MURF-3 polypeptide and a promoter active in said host cell, said promoter directing the expression of said polypeptide, said method comprising culturing the host cell under conditions suitable for the expression of the MURF-1, MURF-2 or MURF-3 polypeptide.
In still yet another aspect of the invention, there is provided an isolated nucleic acid segment comprising at least 15 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5. The isolated nucleic acid segment may be 15, 20, 25, 30, 35, 40, 45, 50, 75 or 100 nucleotides in length. The number of contiguous nucleotides may be increased to 20, 25, 30, 35, 40, 45, 50, 75 or 100.
In still yet an additional embodiment, there is provided as an isolated nucleic acid segment of from 14 to about 888 nucleotides in length that hybridizes to the nucleic acid segment of SEQ ID NO:1, SEQ ID NO:3 SEQ ID NO:5, or complements thereof, under standard hybridization conditions. The isolated nucleic acid segment may further comprise an origin of replication. The isolated nucleic acid may be a viral vector selected from the group consisting of retrovirus, adenovirus, herpesvirus, vaccinia virus, poxvirus, and adeno-associated virus. Further, the isolated nucleic acid may be packaged in a liposome.
In another aspect of the invention, there is provided a nucleic acid detection kit comprising, in suitable container means, an isolated nucleic acid segment that hybridizes under high stringency conditions to the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO:5, or complements thereof. The may further comprise a detection reagent, for example, a detectable label that is linked to said nucleic acid segment.
In yet another embodiment, there is provided a composition comprising a purified MURF-1, MURF-2 or MURF-3 protein or peptide that includes a contiguous amino acid sequence from SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. In still yet another embodiment, there is provided a purified MURF protein having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. In still a further embodiment, there is provided a recombinant MURF-1, MURF-2 or MURF-3 protein or peptide prepared by expressing a DNA segment that encodes a MURF-1, MURF-2 or MURF-3 protein or peptide in a recombinant host cell and purifying the expressed MURF-1, MURF-2 or MURF-3 protein or peptide away from total recombinant host cell components.
In another embodiment, there is provided an isolated peptide of between about 10 and about 50 amino acids in length, comprising a contiguous amino acid sequence from the sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. The peptide may be about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75 or 100 amino acids in length. In yet another embodiment, there is provided an antibody composition that binds to a protein or peptide that includes an epitope from SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. The antibody composition may comprise monoclonal antibodies or polyclonal antibodies. The antibodies of the composition are operatively attached to a detectable label, the label could be selected from the group consisting of a fluorescent label, a chemiluminescent label, a elcetroluminescent label, a radiolabel and an enzyme. Also provided is a hybridoma cell that produces a monoclonal antibody that binds immunologically to MURF-1, MURF-2 or MURF-3. Also provided is an immunodetection kit comprising, in suitable container means, a first antibody that binds to a MURF-1, MURF-2 or MURF-3 protein or peptide.
In still yet another embodiment, there is provided a method for detecting alterations in MURF-1, MURF-2 or MURF-3 function in a cell comprising assessing the structure or expression level of a MURF-1, MURF-2 or MURF-3 polypeptide. The method may comprise determining the structure of a MURF-1, MURF-2 or MURF-3 gene, for example, sequencing a MURF-1, MURF-2 or MURF-3 gene, or Southern or Northern analysis of a MURF-1, MURF-2 or MURF-3 transcript or gene. Alternatively, the assessing may comprise determining the level of a MURF-1, MURF-2 or MURF-3 protein or transcript in the cell, for example, by Northern analysis of MURF-1, MURF-2 or MURF-3 transcripts, or immunodetection of MURF-1, MURF-2 or MURF-3 protein levels (ELISA, Western blot).
In yet a further embodiment, there is provided a method for increasing MURF-1, MURF-2 or MURF-3 activity in cell comprising administering to the cell with an expression construct comprising a MURF-1, MURF-2 or MURF-3 coding region under the control of a promoter active in the cell.
In still yet a further embodiment, there is provided a method of screening a candidate substance for MURF-1, MURF-2 or MURF-3 binding activity comprising (i) providing a MURF-1, MURF-2 or MURF-3 polypeptide; (ii) contacting the MURF-1, MURF-2 or MURF-3 polypeptide with the candidate substance; and (iii) determining the binding of the candidate substance to the MURF-1, MURF-2 or MURF-3 polypeptide. The assay may be performed in a cell free system or in a cell.
In another embodiment, there is provided a method of screening a candidate substance for an effect on MURF-1, MURF-2 or MURF-3 levels in a cell comprising (i) providing a cell that expresses MURF-1, MURF-2 or MURF-3 polypeptide; (ii) contacting the cell with the candidate substance; and (iii) determining the effect of the candidate substance on MURF-1, MURF-2 or MURF-3 polypeptide level.
In still another embodiment, there is provided a method of screening a candidate substance for an effect on MURF-1, MURF-2 or MURF-3 expression in a cell comprising (i) providing a cell that expresses MURF-1, MURF-2 or MURF-3 polypeptide; (ii) contacting the cell with the candidate substance; and (iii) determining the effect of the candidate substance on MURF-1, MURF-2 or MURF-3 mRNA levels.
In still yet another embodiment, there is provided a method of screening a candidate sustance for an effect on MURF-1, MURF-2 or MURF-3 interaction with microtubles comprising (i) providing a microtubule composition; (ii) contacting the microtubule composition with MURF-1, MURF-2 or MURF-3 polypeptide in the presence of the candidate substance; and (iii) assessing the interaction of MURF-1, MURF-2 or MURF-3 with the microtubule composition in the presence of the candidate substance, wherein a change in the interaction of MURF-1, MURF-2 or MURF-3 with the microtubule composition, as compared to the interaction in the absence of the candidate substance, indicates that the candidate substance modulates the interaction of MURF-1, MURF-2 or MURF-3 and microtubules. Step (iii) may comprise a cosedimentation assay.
In another embodiment, there is provided a method for screening a candidate substance for an effect on MURF-1, MURF-2 or MURF-3 homodimeraization comprising (i) providing a MURF-1, MURF-2 or MURF-3 polypeptide composition; (ii) contacting the composition with the candidate substance; and (iii) determining the effect of the candidate substance on MURF-1, MURF-2 or MURF-3 homodimerization.
In still another embodiment, there is provided a method of screening a candidate substance for an effect on MURF-1, MURF-2 or MURF-3 directed glutamic acid modification of microtubules comprising (i) providing a cell that expresses MURF-1, MURF-2 or MURF-3 polypeptide; (ii) contacting the cell with the candidate substance; and (iii) determining the effect of the candidate substance on glutamic acid modification of microtubules.
In yet a further embodiment, there is provided a method of screening a candidate sustance for an effect on MURF-1, MURF-2 or MURF-3 stabilization of microtubles comprising (i) providing a microtubule composition; (ii) contacting the microtubule composition with MURF-1, MURF-2 or MURF-3 polypeptide in the presence of the candidate substance; and (iii) assessing the stability of the microtubule composition in the presence of the candidate substance, wherein a change in the stability of MURF-1, MURF-2 or MURF-3 with the microtubule composition, as compared to the stability in the absence of the candidate substance, indicates that the candidate substance modulates the stability microtubules.
Also provided is a transgenic non-human mammal, cells of which comprise a MURF-1, MURF-2 or MURF-3 encoding nucleic acid segment integrated into their genome, wherein the MURF-1, MURF-2 or MURF-3 encoding nucleic acid is under the control of a heterologous promoter. The promoter may be a tissue specific promoter, for example, a muscle specific promoter, such as myosin light chain-2 promoter, alpha actin promoter, troponin 1 promoter, Naxe2x88x92/Ca2+ exchanger promoter, dystrophin promoter, creatine kinase promoter, alpha7 integrin promoter, brain natriuretic peptide promoter, and alpha B-crystallin/small heat shock protein promoter. The transgenic mammal may be a mouse.
In another embodiment, there is provided a method of treating cardiac failure comprising increasing MURF-1, MURF-2 or MURF-3 activity in a cardiac cell, wherein said increased MURF-1, MURF-2 or MURF-3 activity stabilizes microtubules and/or intermediate filaments. The method may comprise increasing MURF-1, MURF-2 or MURF-3 activity by contacting said cardiac cell with an expression cassette that comprises a polynucleotide encoding a MURF-1, MURF-2 or MURF-3 polypeptide and a promoter active in said cardiac cell, wherein said promoter directing the expression of said polypeptide. The promoter may bea cardiac specific promoter. The contacting may be by intravenous or intraarterial administration of a vector comprising said expression cassette.
In yet a further embodiment, there is provided a method of decreasing MURF-1, MURF-2 or MURF-3 activity in a cell comprising administering to said cell an agent that inhibits MURF-1, MURF-2 and/or MURF-3 activity. The agent be a small molecule, an antisense molecule that hybridizes to MURF-1, MURF-2 and/or MURF-3 transcripts, a ribozyme molecule that cleaves MURF-1, MURF-2 and/or MURF-3 transcripts. Also provided is a method of blocking MURF-1, MURF-2 or MURF-3 expression in a cell comprising administering to said cell an agent that inhibits transcription or translation of MURF-1, MURF-2 and/or MURF-3.
In still a further embodiment, there is provided a method of screening a candidate sustance for an effect on MURF-1, MURF-2 or MURF-3 interaction with intermediate filaments comprising (i) providing an intermediate filament composition; (ii) contacting the intermediate filament composition with MURF-1, MURF-2 or MURF-3 polypeptide in the presence of the candidate substance; and (iii) assessing the interaction of MURF-1, MURF-2 or MURF-3 with the intermediate filament composition in the presence of the candidate substance, wherein a change in the interaction of MURF-1, MURF-2 or MURF-3 with the intermediate filament composition, as compared to the interaction in the absence of the candidate substance, indicates that the candidate substance modulates the interaction of MURF-1, MURF-2 or MURF-3 and intermediate filament. The method may be in a cell or a cell free system. It may be performed in vivo. The method may comprise a cosedimentation assay. The intermediate filaments may be one or more of desmin, vimentin and cytokeratin.
In yet still a further embodiment, there is provided a method for screening a candidate substance for an effect on MURF heterodimerizationcomprising (i) providing two or more of a MURF-1, MURF-2 or MURF-3 polypeptide composition; (ii) contacting the compositions with the candidate substance; and (iii) determining the effect of the candidate substance on the heterodimerization of two or more of MURF-1, MURF-2 or MURF-3.
In each of the preceding screening embodiments, there also is provided similar methods for the production of a modulator, comprising each of the aforementioned screening steps, followed by the additional step of producing the modulator so identified.